PROJECT SUMMARY/ABSTRACT The Problem: Human dental pulps contain mesenchymal stem cells (MSC), i.e. dental pulp stem cells (DPSC) that orchestrate tooth development and tissue regeneration. In the 1st funding cycle, we showed that DPSC are capable of differentiating into endothelial cells that form functional blood vessels. These findings suggest that pluripotent DPSC can differentiate into tissue-forming cells (e.g. odontoblasts) while exhibiting the capacity to generate blood vessels to support the metabolic demands of tissue regeneration. However, the mechanisms employed by DPSC to generate blood vessels are unclear, and therefore our ability to exploit the differentiation potential of these cells in therapeutic tissue regeneration is limited. Hypothesis: We have shown that signaling initiated by vascular endothelial growth factor (VEGF) through its receptor VEGFR1, and by the canonical Wnt/B-catenin pathway, regulate the endothelial differentiation of DPSC. In preliminary studies, we made the exciting observation that DPSC-derived blood vessels anastomize with the host vasculature and become invested by smooth muscle cells/pericytes. However, the mechanisms regulating these events are not known. VE-Cadherin plays a key role in endothelial cell orientation, blood vessel organization and anastomosis during embryonic development. Once blood vessels are formed, the Angiopoietin/Tie2 pathway regulates vessel maturation. Here, we will determine if these signaling pathways play a role in the anastomosis and maturation of post-natal stem cell-derived blood vessels, using dental pulp stem cells as experimental models of MSC. Furthermore, it has been postulated that the long-term viability and function of tissues regenerated with MSC depends on the ability of these cells to reconstitute and maintain stem cell niches. Preliminary data suggest that endothelial cell-derived factors induce self-renewal of DPSC, as demonstrated by Bmi-1 upregulation and secondary sphere formation. Here, we will explore the role of the Stem Cell Factor (SCF)/c-Kit axis on the maintenance of stem cells in DPSC-regenerated tissues. Our working hypothesis is: ?Blood vessels generated by endothelial differentiation of dental pulp stem cells anastomize with the host vasculature, mature upon mural cell investment, and maintain a pool of stem cells?. To test this hypothesis, we propose the following specific aims: S.A.#1: To define the role of VE-Cadherin on the anastomosis of DPSC-derived blood vessels; S.A.#2: To define the function of Angiopoietin signaling on the maturation of DPSC-derived blood vessels; S.A.#3: To define the role of the SCF/c-Kit pathway in the maintenance of stem cells in DPSC-generated tissues. Significance: The clinical translation of stem cell-based therapies requires the understanding of mechanisms that control the differentiation fate of these cells. Successful completion of this project will lead to mechanism- based therapies that exploit the vasculogenic potential of mesenchymal stem cells in tissue regeneration. The development of safe strategies that enable efficient vascularization of tissues generated upon transplantation of stem cells will benefit a large number of patients that require therapeutic tissue regeneration.